Hemoglobin exists in two allosteric forms. The T (taught) and the R (relaxed) form. These forms have different chemical and physical properties and the relative amounts of R and T hemoglobin can be determined by art recognized techniques such as ultraviolet, infrared, visible, nuclear magnetic resonance, and electron spin resonance spectroscopy. For example, Perutz, et al., Biochemistry, 17, No. 17, 3641 (1978) describes absorption spectra of hemoglobin derivatives, i.e., R.fwdarw.T transition as a function of ligand and inositol hexaphosphate binding. Circular dichroism and chemical reactivity are among other techniques for distinguishing R and T states of hemoglobin. The relative amount of R and T states can be determined by both end-point and kinetic techniques.
Elevated levels of glycosylated hemoglobin are known to be associated with diabetes mellitus. Glycosylated hemoglobin is present in nondiabetics at a level of about 5% of total hemoglobin, while diabetics have 2-4 times that amount (Science, 200 April 7, 1978). Glycosylated hemoglobin level provides an index of a patient's average blood glucose concentration over a long period of time. This index is not affected by short-term fluctuations in blood sugar (hour-to-hour) and, hence, gives a relatively precise reflection of the state of blood glucose control in diabetics.
Glycosylated hemoglobin is commonly referred to as HbA, or fast hemoglobin because it migrates faster on a chromatograph column and, indeed, is generally measured by chromatography of electrophoresis.
It has recently (U.S. Ser. No. 973,368 which has a common assignee to this application) been discovered that the percent of glycosylated hemoglobin in blood can be measured by monitoring the shift in the equilibrium populations of R and T allosteric forms of hemoglobins when the nonglycosylated hemoglobin is reacted with an allosteric site binding substance. This reaction causes a shift from the R to the T allosteric form in the nonglycosylated fraction portion of the hemoglobin. The glycosylated hemoglobin in the blood sample does not contribute to the shift in the equilibrium of the allosteric forms since glycosylation blocks the allosteric binding site. Thus, the higher the percentage of glycosylated hemoglobin in the blood sample, the smaller the shift between allosteric forms upon reacting the hemoglobins with an allosteric site binding substance. The earlier discovery takes advantage of the reactivity of the allosteric binding site which is accessible in nonglycosylated hemoglobin and the resulting shift in the equilibrium of allosteric forms of the glycosylated and nonglycosylated hemoglobin mixture resulting when an allosteric binding site substance is reacted with the nonglycosylated hemoglobin fraction.
A wide variety of compounds are known as effective allosteric effector site binding substances, commonly referred to as the organophosphate binding site of hemoglobin. These include organophosphates, sulfates, carboxylic acids represented by inositol hexaphosphate, J. Biol. Chem., 246, 7168 (1971); 2,3-diphosphoglycerate, Nature, 234, 174 (1971); adenosine triphosphate, Biochem. Biophys. Res. Comm., 26, 162 (1967); pyridoxal phosphate, Fed. Proc. Fed. Amer. Soc., Expl. Biol., 28, 604 (1969); inositol pentaphosphate, Can. J. Chem., 47, 63 (1969); 8-hydroxyl-1, 3,6-pyrenetrisulfonate, J. Biol. Chem., 246, 5832 (1971); 0-iodosodium benzoate, The Journal of Pharmacology and Experimental Therapeutics, 203, 72 (1977). Those skilled in the hemoglobin arts will recognize a wide variety of allosteric site binding substances. Inositol hexaphosphate is a preferred allosteric effector site binding substance.
It is generally desirable to lyse red blood cells to release hemoglobins. Common cationic (e.g., cetyl trimethyl ammonium bromide); anionic (e.g., sodium dodecylsulfate and sodium deoxycholate) and neutral (e.g., saponin and octyl phenosypoleythoxyethanol) detergents are useful in lysing red blood cells. Neutral detergents in the concentration range of about 0.025 to 0.5 volume percent are preferred. Mechanical rupture, for example, ultrasonication and hypotonic lysis, are also effective ways of releasing hemoglobin from red blood cells.
Binding of heme-binding ligands to heme iron generally shifts the equilibrium of allosteric hemoglobin isomers to the relaxed (R) form. Thus, when the heme-binding moiety of the hemoglobins in the test sample is coordinated with a heme-binding ligand larger shifts in the equilibrium populations of allosteric forms of hemoglobin are observed. This magnification in shift in equilibrium enhances accuracy and precision of glycosylated hemoglobin determination. This coordination of heme-binding ligand to shift equilibrium of allosteric isomers is applicable when the iron is in the Fe.sup.+2 or the Fe.sup.+3 (methemoglobin) states.
Those skilled in the hemoglobin arts will recognize a wide variety of heme-binding ligands which bind to the iron of hemoglobin or methemoglobin.
For example, isocyanides such as alkyl isocyanides having 1-6 carbon atoms or phenyl isocyanides are particularly desirable heme-binding ligands for hemoglobin in the Fe.sup.+2 state. Other suitable ligands are O.sub.2 and NO.
It is generally preferred to have a single ligand bound to iron since this results in simpler measurements of the shift in allosteric forms. For example, oxyhemoglobin (glycosylated and nonglycosylated) is preferably deoxygenated by reaction with sodium dithionite or other well-known reducing agents to deoxyhemoglobin. The deoxyhemoglobin is reacted with alkylisocyanide such as n-butylisocyanide and as a result reaction with an allosteric effector site binding ligand provides a more definite shift in equilibrium of the allosteric forms permitting determination of glycosylated hemoglobin.
Hemoglobin is oxidized to methemoglobin by art recognized techniques, Antonini and Brunoni, Hemoglobin and Myoglobin in Their Reaction With Ligands, North Holland Publishing Co., Amsterdam (1971). Thus, potassium ferricyanide, sodium nitrite, aniline, and phenylhydrazine are convenient reagents for oxidizing hemoglobin to methemoglobin. Autooxidation in the presence of dyes such as methylene blue also oxidizes hemoglobin to methemoglobin.
Nonglycosylated methemoglobin is reactive with allosteric effector site binding substances described for nonglycosylated hemoglobin.
Those skilled in the hemoglobin arts will recognize a large variety of heme-binding ligands which bind with methemoglobin. These ligands include cyanate, thiocyanate, N-hydroxyacetamide, imidazole and derivatives thereof. Perutz, et al., Biochemistry, 17, 3640-3652 (1978).
Other common ligands are fluoride, azide, nitrite, cyanide, water, hydroxide ammonia, acetate and formate. Imidazole at about 0.1 M is a preferred heme-binding ligand for use with methemoglobin.
Typically, 1 ml of a reagent which is 0.1 M imidazole, 0.2 mM potassium ferricyanide, K.sub.3 Fe(CN).sub.6, and 0.05% by volume triton X-100 (octyl phenoxypolyethoxyethanol) detergent in buffer at pH 6.8 is added to 10-20 .mu.l of whole blood and the mixture is incubated for ten minutes.
The potassium ferricyanide oxidizes the hemoglobin to methemoglobin; the triton X-100 is a neutral detergent which lyses the cells to release hemoglobins; and the imidazole coordinates with the iron shifting equilibrium allosteric isomers to the (R) form.
The absorption spectrum of this mixture is recorded at 560 nm and 635 nm. Then 2 .mu.l of a 0.1 M inositol hexaphosphate solution, pH 6.8 is added. The latter reagent reacts with the allosteric binding site of nonglycosylated hemoglobin and shifts equilibrium of the allosteric isomers to the (T) target form. The absorption spectrum at 560 nm and 635 nm is measured again. Glycosylated hemoglobin concentration is reflected by a decrease in 560 nm absorption and increased in the 635 nm absorption.
Reagent A: 0.1M imidazole, .2 mM K.sub.3 Fe(CN).sub.6, 0.05% v/v triton X-100 (octyl phenoxypolyethoxyethanol detergent), in water, pH 6.8
Reagent B: 0.1 M inositol hexaphospate (IHP), in water, pH 6.8
To 1.0 ml of Reagent A at 25.degree. C. add 10-20 .mu.l whole blood, incubate 10 minutes to allow for cell lysis and oxidation of hemoglobin to methemoglobin. Record visible spectrum, 450 nm to 700 nm, specifically monitoring absorbance at 560 nm and 635 nm. Then add 2 .mu.l Reagent B to the reaction mixture. Record another spectrum as before.
Standards prepared by methods of this invention are used to determine glycosylated hemoglobin present.
______________________________________ RESULTS Standard Curve Normalized No IHP + IHP Difference % Glycosylated Hb 560nm A 635nm A 560nm A 635nm A ##STR1## ______________________________________ 0% 0.664 0.089 0.592 0.123 0.184 5% 0.654 0.086 0.588 0.120 0.176 10% 0.657 0.089 0.593 0.121 0.169 15% 0.658 0.090 0.596 0.118 0.158 20% 0.663 0.095 0.609 0.123 0.144 25% 0.651 0.091 0.600 0.117 0.138 50% 0.645 0.098 0.611 0.113 0.090 100% 0.717 0.123 0.715 0.128 0.012 ______________________________________ Calculations: .DELTA. = A.sup.560nm - A.sup.635nm ##STR2## -